The present invention concerns class II cytochrome P450s, electrochemical systems for assaying cytochrome P450 catalytic activity, apparatus and methods for same.
Cytochromes P450 constitute a large family of haem thiolate enzymes, found in bacteria, fungi, plants, insects and animals, which catalyse a wide range of reactions, including hydroxylation of aliphatic and aromatic carbons, epoxidation, oxidative deamination, sulphoxide formation, N-oxidation, N-hydroxylation, dehalogenation and N-, O- and S-dealkylation. These diverse reactions all arise from the fact that cytochromes P450 are versatile oxygen-activation catalysts which incorporate one atom of molecular oxygen into a broad range of substrates with concomitant reduction of the other oxygen atom to water. These enzymes play important roles in the biosynthesis of secondary metabolites in plants, in steroid metabolism in fungi and animals and, notably, in xenobiotic metabolism.
The considerable diversity of P450s results in man being able to metabolize a wide range of foreign chemicals. Metabolism by P450s has a major influence on the pharmaco-toxicological properties of therapeutic drugs, and consideration of this metabolism is a key part of the drug design process. Furthermore, several P450s are polymorphic in the human population, resulting in individual differences in drug and toxin metabolism. In a different sphere, the ability of cytochromes P450 to hydroxylate unactivated carbons suggests that they have potential applications in synthetic chemistry.
In vivo, P450s require other proteins as electron donors. The class I enzymes (most bacterial P450s, and the mitochondrial steroid-metabolising enzymes) require an NADH-dependent reductase and an iron-sulphur protein, while the class II enzymes (e.g. the mammalian drug-metabolising enzymes) simply require a flavoprotein NADPH-dependent reductase. The mammalian drug-metabolising mono-oxygenase system thus consists of NADPH-cytochrome P450 reductase and a number of different P450s, all of which are bound to the membranes of the endoplasmic reticulum. (By contrast, the only known bacterial class II P450, P450 BM3 (CYP102) from Bacillus megaterium, is a soluble enzyme having both P450 and NADPH-cytochrome P450 reductase activities in a single polypeptide chain.)
The assay of cytochromes P450, for example to study the metabolism of a new therapeutic agent, has classically involved incubation of the candidate substrate with a microsomal membrane preparation from the cells of interest, together with NADPH as an electron source, followed by chromatographic analysis for product formation. Because several different P450s are expressed in most cell types, it is difficult in this kind of assay to identify the specific P450(s) responsible for metabolism of the compound of interest.
More recently, therefore, individual recombinant P450s expressed in bacteria, yeast, insect or mammalian cells (see, for example Gonzalez, F. J. et al., 1995, Ann. Rev. Pharmacol. Toxicol., 36: 369-390; Pritchard, M. P. et al., 1997, Arch. Biochem. Biophys. 345: 342-354; Guengerich, F. P. et al., 1997, Curr. Opin. Biotechnol., 8: 623-628) have been used in combination with endogenous or recombinant P450 reductase. These assays have generally also involved chromatographic analysis; while NADPH consumption can be followed spectrophotometrically, the coupling between electron flow and product formation is variable and hence NADPH consumption may not be a reliable indicator of metabolism of a given compound.
It has recently been shown that electrons can be supplied to P450 reductase electrochemically from a platinum electrode by means of a mediator, cobalt sepulchrate (Estabrook, R. W. et al., Meth. Enzymol., 272: 44-51), and on the basis of this systems have been developed in which fusion proteins comprising NADPH-cytochrome P450 reductase and a class II cytochrome P450 can be driven electrochemically (Estabrook, R. W. et al, 1996, Endocrine Res., 22: 665-671). In the case of the quite different class I P450s, it has recently been shown that electrons can be supplied electrochemically either via putidaredoxin (Reipa, V. et al., 1997, PNAS USA, 94: 13554-13558) or directly to the P450 if the latter is immobilised in a lipid film (Zhang, Z. et al., J. Chem. Soc. Faraday Trans., 93; 1769-1774).
Further examples of prior art systems involving the use of Class I P450 enzymes such as P450cam are given in Kazlauskaite, J. et al. (1996, Chem. Commun (Cambridge), 18: 2189-2190), Vilker, V. L. et al. (Redox Chem. Interfacial Behav. Biol. Mol., 1987, 105-112), Vilker, V. L. et al. (1997, Proc. Electrochem. Soc., Volume 97-6: 91-99) and GB 2312960.
The present invention overcomes the prior art disadvantages, particularly those associated with the use of a P450 reductase to supply electrons to a P450, and provides simple and convenient methods and arrangements for supplying electrons to class II cytochromes P450, particularly for assaying P450 catalytic activity.
Although it has not previously been possible to supply electrons to P450 cytochromes without the use of P450 reductases, the present invention obviates this need.
Thus according to the present invention there is provided a class II cytochrome P450 attached to a graphite electrode. In contrast to the prior art this attachment does not require P450 reductase and thus provides a substantial advantage over the prior art.
The P450 cytochrome is attached such that upon catalysis of a reaction by the enzyme, electron flow occurs from the electrode to the enzyme.
The P450 cytochrome may be attached to the electrode via dodecyl dimethylammonium bromide (DDAB). It may be attached via other amphiphilic molecules which are insoluble in aqueous solutions. Such molecules are well known and will be readily apparent to one skilled in the art. They include Nafion (RTM) and phosphatidylcholine.
Also provided according to the present invention is apparatus for determining the catalytic activity of a class II P450 cytochrome, comprising a cell having a graphite electrode having attached to it the cytochrome P450, and also having electron flow detection means.
The inventors have also found that it is not necessary for the P450 cytochrome to be bound to an electrode, and thus also provided according to the present invention is apparatus for detecting the catalytic activity of a class II P450 cytochrome comprising a cell having a modified gold electrode contacting a solution containing the enzyme.
The gold electrode may be coated in 2,2-dithiodipyridine (Adrithiol) or by other polyfunctional molecules which can be adsorbed onto the electrode and also interact specifically with the cytochrome P450. These molecules will be readily apparent to one skilled in the art, and include those compounds disclosed as xe2x80x9cmediatorsxe2x80x9d by Christensen, P. A. and Hamnett, A. (xe2x80x9cTechniques and Mechanisms in Electrochemistryxe2x80x9d, 1994, Blackwell Academic Press, London, pp. 356-373) which is incorporated herein by reference in its entireity. The polyfunctional molecules may have the structure ofxe2x80x9cType IVxe2x80x9d compounds discussed by Christensen and Hamnett (supra) and Allen, P. M. et al., 1984, J. Electroanal. Chem., 178: 69 which is incorporated herein by reference in its entireity . The compounds may include 1,2 bis(4-pyridyl) ethylene, 4,4xe2x80x2 bipyridine, bis (4-pyridyl) bisulphide, or 4 mercaptopyridine. Such modified gold electrodes are able to supply electrons to the cytochrome P450 yet do not need to be attached by strong chemical bonds to the cytochrome P450.
Compounds suitable to act as mediators of electron flow from and electrode to an enzyme are also disclosed by R. W. Murray (Acc. Chem. Res., 1980, 13, 135) which is incorporated in its entireity herein by reference.
Generally speaking, cells may be three-electrode cells having a first electrode as described above (i.e. graphite or gold) and second and third electrodes comprising a saturated calomel working electrode and a platinum wire counter-electrode. Naturally, each electrode should contact the solution to be tested for the presence of a substrate for the P450 cytochrome.
The electron flow detection means may monitor current flow or may determine a steady state cyclic voltammogram, for example over the range xe2x88x920.4V to +0.4V.
The cytochrome P450 catalytic activity may also result in the generation of peroxides, essentially a side-product when it is desired to assay the generation of a specific product resulting from catalysis. The generation of peroxides results in electron flow and thus affects the results obtained by the apparatus. Thus the apparatus may additionally comprise means for detecting peroxide formation. Such means are well known and include colourimetric peroxide assay means and fluorimetric peroxide assay means. Alternatively, it may comprise an electrode which detects peroxide. Thus the electron flow required for the assayed peroxide generation can be subtracted from the total electron flow and a more accurate measure of product formation obtained.
A range of applications exists for the present invention, particularly for the screening of novel compounds as substrates to a cytochrome P450 and for the use of P450 catalysis in synthetic chemistry. For example a high-throughput screening system for P450 substrates could be readily created. In the case of using P450s for synthetic chemistry, the ability of P450s to catalyse hydroxylation of unactivated carbons, together with their broad substrate specificity, makes them attractive tools for synthetic chemistry. However, the high cost of NADPH has to date hampered this. The ability of the present invention to drive reactions electrochemically now makes their use more commercially attractive.
Thus the present invention also provides a method of determining the catalytic activity of a class II cytichrime P450, comprising the use of apparatus according to the present invention. It may be a method for determining whether a given compound is a substrate for a class II cytochrome P450.
Also provided is a method of performing a synthetic chemical reaction, comprising the use of apparatus according to the present invention.